PROJECT SUMMARY Multiple Sclerosis (MS) is the most common neurological disease of early adulthood and is mediated by autoimmune mechanisms that lead to demyelination and neuronal damage in the central nervous system, resulting in progressive neurological dysfunction. There is no cure for the disease and current treatments focus on preventing future immunological attacks, mainly by suppressing the immune system. This leads to adverse side effects that are often severe or fatal. Accordingly, there is a clear unmet need for the development of effective and well-tolerated therapies to arrest MS development. This has been challenging because MS has multiple etiologies and the molecular mechanisms underlying these etiologies are not well understood. We uncovered the molecular underpinnings of an MS etiology and hope this knowledge will translate into an accurate therapy for MS. This etiology is associated with the interleukin 7 receptor (IL7R) gene, which encodes a cell surface receptor in T cells (hereafter referred to as mIL7R) that plays a central role in the homeostasis of T cells. We previously identified the genetic variant rs6897932 within exon 6 of IL7R to be strongly associated with increased MS risk, and showed the risk allele of this variant increases exclusion of the alternative exon 6 leading to higher expression of mRNAs encoding a secreted form of the receptor (sIL7R) and elevated levels of circulating sIL7R. This has important implications in the development of MS because sIL7R has been shown to aggravate the progression and severity of the disease in the Experimental Autoimmune Encephalomyelitis (EAE) mouse model of MS. Further supporting this, elevated levels of sIL7R have been reported in patients of several autoimmune diseases including MS, Type I diabetes, Rheumatoid arthritis and Systemic lupus erythematosus. Given that sIL7R is produced by exclusion of exon 6 during alternative splicing of IL7R RNAs, we developed a novel biologic drug, a splicing-modulating antisense oligonucleotide (SM-ASO) that corrects splicing of IL7R exon 6 and restores normal expression of IL7R protein isoforms. Our approach represents a major improvement over current MS therapies in that by correcting IL7R splicing, it diminishes expression of the pathogenic sIL7R isoform, without reducing expression of the mIL7R. This is important because mIL7R function is vital for proper immune function and its disruption leads to immunodeficiency. Thus our biologic drug, unlike current MS drugs, will not cause immunosuppression. Because IL7R expression is mostly restricted to T cells, these are the major producers of sIL7R and thus the main target of our IL7R SM-ASOs. Although SM-ASOs have been shown to modulate RNA splicing decisions in many cell types in vitro and in vivo (e.g. FDA-approved Spinraza), the delivery and functionality of SM-ASOs in T cells have not been thoroughly examined, and represents the major hurdle to expand the use of SM-ASOs for treatment of immunological disorders and immunotherapies. Here, we address this obstacle by testing the influence of diverse chemical modifications on the efficiency of delivery and activity of ASOs in primary T cells.